DESCRIPTION: The principal investigator will study human SH-SY-5Y neuroblastoma cells which possess m3 muscarinic cholinergic receptors linked to phospholipase C activation. These cells will be used to study molecular mechanisms by which cell surface receptors become internalized upon continuous agonist occupancy. He will focus upon the hypothesis that mAChRs are internalized via clathrin coated pits by an ATP-dependent mechanism. Three hypotheses are to be tested: 1) Muscarinic receptors are sequestered by a pathway that is common to that utilized by constitutively active receptors. Does sequestration involve a clathrin coated pit mechanism? To test this, procedures known to disrupt formation or budding of clathrin coated pits will be introduced. 2) Internalization of the muscarinic receptors requires ATP and cytosol and is regulated by G proteins. 3) Muscarinic receptors are sequestered into an environment deficient in the key components of the PPI signaling pathway and in substrate availability.